Title: Development of catch and release approach for multi-drug local delivery of chemotherapeutics. Project Summary: The goal of this proposal is to establish a new transformative paradigm for local tumor control, independent of molecular markers or metabolic activity, that allows tailored multi-drug dosing regimens. The project addresses the increasing need of developing new treatments of pediatric sarcomas. The five-year survival rates of patients with this ailment are at 67% and have not improved since the 1990's. There have been no major therapeutic improvements for the remaining third of patients in over 25 years. Meanwhile, only 25% of 120 new cancer drugs approved by the FDA between 1948 and 2002 are used in children. The proposed materials-based strategy termed `catch and release' is based on bio-orthogonal inverse-electron demand Diels-Alder (IEDDA) reaction between tetrazine and trans-cyclooctene (TCO). The key element of the proposed design is biocompatible hydrogel, modified with tetrazine (HMT), injected in the vicinity of a local sarcoma tumor. Pro-drugs with attenuated activity and minimal side effects, containing a releasable TCO moiety will be systemically injected. When the pro-drug and the hydrogel come in contact, the bio-orthogonal agents react with each other through IEDDA reaction `catching' the payload. Finally, the resulting intermediate isomerizes spontaneously releasing the active cytotoxic compound from the hydrogel to perform its therapeutic function locally. Preliminary in vitro data has shown that HMT is stable under simulated physiological conditions and capable of activating a pro-drug of doxorubicin. Meanwhile, preliminary in vivo testing proved that the `catch and release' strategy is capable of local activation of therapeutically meaningful quantities of doxorubicin to treat sarcoma. Multivalency of HMT allows for the process to be repeated with multiple doses of the systemically administered pro-drugs. The proposed project will focus on the ability of HMT for local activation of multiple doses of different chemotherapeutic drugs. Specific aim 1 will focus on the synthesis, as well as in vitro characterization of cytotoxicity of the pro-drugs of doxorubicin and etoposide in combination and as a mono-therapy. Kinetics of in vitro activation by HMT will also be tested. The Specific aim 2 will be a 3-round single dose up-and-down study to determine the maximum tolerated dose (MTD) and serum pharmacokinetic behavior. MTDs will also be determined for combinations of the Aim 1 pro-drugs after administration of a single intravenous dose in rats. During Specific Aim 3, we will evaluate the therapeutic response to mono-therapy and combination pro-drug treatment in a range of sarcoma types using patient-derived xenograft (PDX) sarcoma model in mice ? 2 models of osteosarcoma, and 1 model each of rhabdomyosarcoma and Ewing's sarcoma. In the long term, our goal is to develop a new materials-based approach for treatment of local tumors that will have lower side- effects and minimally alter the regular cancer treatment workflow.